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Objective To elucidate activity of baicalin against human cytomegalovirus ( HCMV) in vitro, and explore its effect on apoptosis of human embryo lung fibroblasts ( HEL ) infected with HCMV. Methods CCK-8 kit was used to determine the maximum tolerated dose ( MTC ) of HEL cell to baicalin while the anti-HCMV median efficacious concentration ( EC50 ) of baicalin was determined by standard plaque reduction method. After treatment with baicalin of different concentrations for 24, 48, 72 and 96 h, cell apoptosis and pro-caspase-3 expression was detected by flow cytometry and Western blotting, respectively. Results The MTC of baicalin was 20.6 μg?mL-1; The EC50 of anti-HCMV of baicalin was 16.13 μg?mL-1;The apoptosis rate increased gradually in the groups with low and high multiplicity HCMV infection at early time, showing significant dose-dependent manner. While the ratio of apoptotic cells was going to decrease in high multiplicity infection group 96 h after the infection. The expression of pro-caspase-3 was significantly higher in high-dose baicalin treatment group than in the infection control group ( P<0.05) . Conclusion Baicalin has a direct anti-HCMV effect in vitro. One of the mechanisms might be related with it inhibiting cell apoptosis and antagonizing activation of pro-caspase-3.
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Objective To elucidate activity of baicalin against human cytomegalovirus ( HCMV) in vitro, and explore its effect on apoptosis of human embryo lung fibroblasts ( HEL ) infected with HCMV. Methods CCK-8 kit was used to determine the maximum tolerated dose ( MTC ) of HEL cell to baicalin while the anti-HCMV median efficacious concentration ( EC50 ) of baicalin was determined by standard plaque reduction method. After treatment with baicalin of different concentrations for 24, 48, 72 and 96 h, cell apoptosis and pro-caspase-3 expression was detected by flow cytometry and Western blotting, respectively. Results The MTC of baicalin was 20.6 μg?mL-1; The EC50 of anti-HCMV of baicalin was 16.13 μg?mL-1;The apoptosis rate increased gradually in the groups with low and high multiplicity HCMV infection at early time, showing significant dose-dependent manner. While the ratio of apoptotic cells was going to decrease in high multiplicity infection group 96 h after the infection. The expression of pro-caspase-3 was significantly higher in high-dose baicalin treatment group than in the infection control group ( P<0.05) . Conclusion Baicalin has a direct anti-HCMV effect in vitro. One of the mechanisms might be related with it inhibiting cell apoptosis and antagonizing activation of pro-caspase-3.
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Objective:To construct the pEGFP-C2-hKGF vector,a neukaryotic expression plasmid of hKGF gene in order to provide experimental material for the studies of hKGF gene function and hKGF gene therapy afterward.Methods:The hKGF gene was amplified by reverse transcriptage polymerase chain reaction(RT-PCR).Total RNA was extracted from human embryo lung fibroblast(HLF)and then cloned into pMD18-T vector.The recombinant plasmid were screened and extracted.The pEGFP-C2-hKGF vector,an eukaryotic expression plasmid of hKGF gene was constructed after the object hKGF cDNA fragment was inserted into pEGFP-C2 vector and the recombinant were screened,cloned and extracted.The recombinant plasmid vector was transferred into AEC II,and expression of hKGF was assayed by immunohistochemistry.The effect of AEC II proliferation was analyzed by cell count.Results:609bp DNA fragment containing hKGF gene and restriction sites were obtained by RT-PCR.After cloned by pMD18-T vector and certified by restriction analysis and sequencing,pEGFP-C2-hKGF vector was successfully constructed by means of recombinant DNA technology.pEGFP-C2-hKGF encoding the green fluorescent protein(GFP),capable of directing the expression of functional hKGF successfully.Furthermore,hKGF had the effect on AEC II proliferation.Conclusion:The efficient expression vector of hKGF gene is constructed,which can be used to establish the hKGF gene therapy vector.
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Objective To study the DNA damage and repair of normal lung interstitial cells and human lung adenocarcinoma cells exposed to cigarette smoke. Methods Cultured human embryo lung fibroblasts (HLF) and human lung adenocarcinoma A549 cells. Mainstream smoke was collected by using dimerhyl sulfoxide (DMSO) and phosphate buffer solution (PBS) as absorbents. MTT assay was used to test the cytotoxicity of the solutions of cigarette smoke, then selected the concentrations of the solutions with no obvious cytotoxicity to treat cells and detected DNA damage and repair by comet assay. Results As treated with original solutions or 1/2 dilution of DMSO cigarette smoke solutions only, the Viability of cells was below 80%, but it was beyond 80% when treated with PBS solutions. The results showed that a significant difference of DNA damage was seen between the treated groups and negative control groups (P0.05),but the DNA damage caused by DMSO solutions was worse than PBS solutions significantly (P